High throughput screening method and apparatus

ABSTRACT

High-throughput screening method and apparatus arm described. The method includes placing cells on a substrate defining a plurality of discrete microwells, at a well density of greater than about 100/cm 2 , with the number of cells in each well being less than about 1000, and where the cells in each well have been exposed to a selected agent. The change in conductance in each well is determined by applying a low-voltage, AC signal across a pair of electrodes placed in that well, and synchronously measuring the conductance across the electrodes, to monitor the level of growth or metabolic activity of cells contained in each well. Also disclosed is an apparatus for carrying out the screening method.

FIELD OF THE INVENTION

[0001] The present invention relates to high throughput screening (HTS)methods, e.g., for detecting the effect of a given compound or treatmenton cell metabolic activity, and apparatus for performing such screening.

BACKGROUND OF THE INVENTION

[0002] With the advent of combinatorial library methods for generatinglarge libraries of compounds, there has been a growing interest inhigh-throughput screening (HTS) methods for screening such libraries.

[0003] The most widely used HTS screening method involves competitive ornon-competitive binding of library compounds to a selected targetprotein, such as an antibody or receptor. Thus, for example, to select alibrary compound capable of blocking the binding of a selected agonistto a receptor protein, the screening method could assay for the abilityof library compounds to displace radio-labeled agonist from the targetprotein.

[0004] Although such binding assays can be used to rapidly screen largenumbers of compounds for a selected binding activity, the assay itselfmay have limited relevance to the actual biological activity of thecompound in vivo, e.g., its ability to interact with and affect themetabolic behavior of a target cell.

[0005] It would therefore be useful to provide high throughput screeningmethods capable of testing the effects of large numbers of librarycompounds on target cells of interest.

SUMMARY OF THE INVENTION

[0006] The invention includes, in one aspect, high throughput screeningapparatus, e.g., for screening the effect of test compounds on cellmetabolic activity, or for screening the effect of a geneticmanipulations on cells. The apparatus includes a multiwell devicedefining a plurality of discrete microwells on a substrate surface, at awell density of greater than about 100/cm², where the well volumes aresuch as to accommodate at most about 10⁶ cells/well, preferably between1-100 wells/cell, and structure for measuring the conductance in eachwell. The measuring structure includes (i) a pair of electrodes adaptedfor insertion into a well on the substrate, and (ii) circuitry forapplying a low-voltage, AC signal across the electrodes, when theelectrodes are submerged in the medium, and for synchronously measuringthe current across the electrodes, to monitor the level of growth ormetabolic activity of cells contained in the chamber.

[0007] In various preferred embodiments. the signal circuitry iseffective to generate a signal whose peak-to-peak voltage is between 5and 10 mV, and includes feedback means for adjusting the signal voltagelevel to a selected peak-to-peak voltage between 5 and 10 mV.

[0008] In other embodiments, the circuitry is designed to sample thevoltage of the applied signal at a selected phase angle of the signal,or alternatively, to sample the voltage of the applied signal at afrequency which is at least an order of magnitude greater than that ofthe signal.

[0009] In another general aspect, the invention includes ahigh-throughput screening method, e.g., for screening the effect of testcompounds on cell metabolic activity, or the effect of a given geneticmanipulation. The method includes placing cells in the wells of amultiwell device defining a plurality of discrete microwells on asubstrate surface, at a well density of greater than about 100/cm², withthe number of cells in each well being less than about 10⁶, andpreferably between 1-10³. The conductance in each well is determined byapplying a low-voltage, AC signal across a pair of electrodes placed inthat well, and synchronously measuring the conductance across theelectrodes.

[0010] These and other objects and features of the invention will becomemore fully apparent when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a perspective view of a multiwell device that forms partof the apparatus of the invention;

[0012]FIG. 2A is an enlarged, fragmentary cross-sectional view of thedevice in FIG. 1, with the wells in the device each containing a smallnumber of cells suspended in a culture medium;

[0013]FIG. 2B is a view like that in FIG. 2A, but further showing anelectrode cover on the device;

[0014]FIG. 3 illustrates the grid of conductive wires in the electrodeplate shown in FIG. 2B;

[0015] FIGS. 4A-4C illustrate another embodiment of the apparatus, heredesigned for well-by-well conductance measurements;

[0016]FIG. 5 shows idealized plots of conductance, as a function oftime, in the presence and absence of a library compound that inhibitscell growth or metabolism;

[0017]FIG. 6 is a block diagram depicting selected portions of the dataacquisition board and of the measurement input/output board inaccordance with the present invention; and

[0018]FIG. 7 depicts waveforms for the alternating current voltagesupplied for application across a pair of pins, an output signalproduced by a comparator in response to the alternating current voltage,and a hypothetical output signal from a sample-and-hold amplifier.

DETAILED DESCRIPTION OF THE INVENTION

[0019] A. Screening Apparatus and Method

[0020]FIG. 1 shows a perspective view of a multiwell device X formed ofa substrate 10 having a plurality of microwells, such as wells 12, onthe upper substrate surface 14. In the embodiment shown, the microwellsare formed by a grid of hydrophobic lines, such as lines 16 extendinglengthwise, and lines 18 extending widthwise. The lines are preferablyformed of a hydrophobic polymer material, such as polyethylene orpolystyrene, and are laid down in a conventional manner, e.g.,deposition of melted polymer from an applicator, or heat-mediatedattachment of a polymer grid fabric directly to the substrate surface.

[0021] Spacing between adjacent parallel lines is preferably 20-200 μm,so that the wells formed by intersecting lines have area dimensions ofbetween about 400 to 40,000 μm². The density of wells on the substrateis at least 100/cm² and more preferably 10³/cm² to 10⁴/cm or greater.

[0022] The height (surface relief) of the grid lines, seen best in FIGS.2B and 2B, is typically between 20-200 μm. Microwell volumes, defined bythe volume that can be held as a discrete droplet in a microwell, aretypically in the range 10⁻⁴ to 2 nanoliters.

[0023] The microwells in the device may be filled with selectedbiological cells by applying a suspension of the cells, at a desiredcell density, over the device's surface, and allowing excess suspensionfluid to drain off, e.g., by blotting the edges of the device. This isillustrated in FIGS. 2A and 2B which show cell suspension droplets, suchas droplet 20, in the microwells of the device, such as microwell 22. Itwill be appreciated that the droplet meniscus may extend above theheight of the grid lines.

[0024] The cell density is adjusted so that the wells are filled, onaverage, with a selected number of cells which is preferably between1-100, but may be as high as 10³ per well. As illustrated in FIGS. 2Aand 2B, the density of the cells is such that the device has an averageof about 2 cells/well. A greater number of cells/well, e.g., an averageof 10-100/well provides improved statistical correlation among eventsobserved in different cells, due to more uniform cell-numberdistribution in the microwells of the device. At the same time, a smallnumber of cells, e.g., 10-100, allows a microwell (microvolume) formatin which desired concentrations of test compounds can be achieved withvery small amounts of compound, e.g., in the femptogram to nanogramrange. The actual number of cells employed will depend on the particulartype of cell and medium, and the confluency requirements of the cells.

[0025] With reference particularly to FIG. 2B, the apparatus of theinvention, further includes means for measuring the conductance of thecell medium in each well. In the embodiment shown, this means includes amulti-electrode cover 27 having a plurality of electrodes pairs, such aselectrode pair 26 made up of electrodes 26 a, 26 b formed as a grid onthe lower side of the cover. Specifically, the grid of electrode pairsmatches that of microwells in device 8, so that placement of the coveron the device places an electrode pair in each microwell.

[0026] The measuring means also includes a signal unit 28 electricallyconnected to the cover as described below with reference to FIG. 3. Theoperation of the unit to provide a low-voltage AC signal to eachelectrode, and interrogate the electrode to determine the conductance ineach microwell is described below with reference to FIGS. 6 and 7.

[0027]FIG. 3 is a plan view of cover 27, showing the grid pattern ofconductive wires, such as longitudinal wires 30, and lateral wires 32,connecting the electrode pairs in the cover. Each longitudinal wire isconnected to an electrode connector, such as wires 32 connected toconnectors 34, in a multi-connector array 36 along one side of thecover, as shown. Similarly, each lateral wire is connected to anelectrode connector, such as wires 32 connected to connectors 38, in amulti-connector array 40 along another side of the cover. The two arraysare designed to plug into matching ports in the signal unit.

[0028] As seen in FIG. 2B, each longitudinal wire, such as wire 30, iselectrically connected to one of the two electrodes in the longitudinalone-dimensional array of electrode pairs, such as the array includingpair 26, adjacent the wire. Similarly, each lateral wire, such as wire36, is electrically connected to one of the two electrodes in thelateral one-dimensional array of electrode pairs, such as the arrayincluding pair 26, adjacent that wire.

[0029] Thus, to interrogate a particular microwell in the device, thesignal unit applies a low-voltage signal across the two connectors incover 27 which are connected to the two electrodes in that well. Forexample, to interrogate microwell 26 in FIG. 2B, the signal unit appliesa voltage signal across connectors 34, 38 connected to electrodes 26 a,26 b, respectively, forming the electrode pair in that microwell. Tothis end, the signal unit includes the basic electronics for applying alow-voltage signal to the electrodes, and for synchronously measuringthe current across the electrodes, as described below.

[0030] Unit 28 also includes conventional multiplexing or samplingcircuitry for alternately and successively interrogating each microwell,by applying a short duration signal to successively to each well, andmeasuring the current across the “stimulated” electrodes in accordancewith the signalling and current measuring procedures described below.According to one feature of the signal unit, the time requiredaccurately interrogate each microwell can be quite short, on the orderof only a few cycles on the applied signal, allowing large arrays to becontinuously monitored in real time.

[0031] FIGS. 4A-4C illustrate a HTS apparatus 42 constructed accordingto another embodiment of the invention. The apparatus includes amultiwell device 44 similar to above described device 8, and having aplanar array of microwells, such as wells 46, 48. In this embodiment,however, the measuring means for determining the conductance of eachwell is carried out by an electrode arm 50 having a pair of electrodes52, 54 adapted to be received in a selected microwell of the device, andconnected to a signal unit 56. The electrode arm is movable in the “z”plane between raised and lowered positions in which the electrodes areposition above, and in a selected microwell, respectively, asillustrated in FIGS. 4A and 4B, respectively. This movement is producedby a vertical actuator, indicated by arrow 58, which is also under thecontrol of unit 50.

[0032] Also forming part of the apparatus is a stage 60 on which thedevice is placed during operation. The stage is movable, in an “x-y”plane under the control of signal unit 56, to alternatively andsuccessively bring each microwell in the device to an interrogationposition directly below the electrode arm, as indicated for well 46 inFIGS. 4A and 4B. The signal unit also includes the basic electronics forapplying a low-voltage signal to the electrodes, and for synchronouslymeasuring the current across the electrodes, as described below.

[0033] In an exemplary operation, for use in screening combinatoriallibrary compounds, and with reference particularly to the embodimentshown in FIGS. 1-3, the microwells in the device are filled with a cellsuspension, as above, and the library compounds are added to each ofwells, either before or after cell addition. For example, usingmicrofabrication techniques of the type described in U.S. Pat. No.5,143,854, a position-addressable planar array of polymer librarymolecules is formed on a planar substrate—in this case having orsubsequently prepared to have a hydrophobic grid forming the microwells,which correspond to the individual library-polymer regions. Afteraddition of the cells, the library compounds may be released forinteraction with the cells, e.g., by inclusion in the cell suspension ofan enzyme capable of cleaving the library molecules from the substratesurface.

[0034] Alternatively, the library compounds may be contained on a gridof pins or the like corresponding to the microwell grid, allowing thecompounds to be simultaneously introduced into the wells, and releasedinto the corresponding wells, e.g., by enzymatic cleavage of a linker.

[0035] Alternatively, the library compounds may be distributedwell-by-well into the microwell device, either as single compounds ormixtures of library compounds.

[0036] After introducing the compound to be tested, the microwells areinterrogated to determine the conductance of medium in each cell, bymeasuring the current across the electrodes. In the present example, itis assumed that (i) a large number of individual library compounds areadded to the device. one per well, and (ii) one or more of the compoundsis able to inhibit metabolic activity and/or replication of the cells.In the absence of any inhibition, cell metabolism and growth will occurnormally, leading to an increase in measured conductance over time, asillustrated by plot number 1 in FIG. 5. Since only a few of the testcompounds will be expected to have an inhibitory effect, most or nearlyall of the plots will be represented by the “normal” plot.

[0037] Where an inhibitory compound is present, this will be evidencedby a lower rate of conductance change over time, as indicated by curves2 and 3 in FIG. 5, where curve 2 represents moderate inhibition, andcurve 3, nearly complete inhibition. The reduced conductance may be dueto reduced metabolism and/or reduced replication. To confirm the latterpossibility, the microwells of interest may be further examined for cellcount, using standard cell counting methods.

[0038] In another general embodiment, the screening method is employedto monitor the success of a selected genetic manipulation, e.g.,transformation of the cells with a selected vector, treatment with atransforming virus such as EBV, or cell fusion. In this embodiment, thegenetically manipulated cells are distributed on the microwell device asabove, and changes in cell conductance, related to cell replication aremonitored. Those wells that show significant increase in cellconductance over time are then selected as cells which are successfullymanipulated. Thus, for example, if the cells are transformed with avector containing a selectable antibiotic marker gene, cells which growin the presence of the antibiotic can be readily identified by theincreased conductance in the corresponding microwell(s).

[0039] B. Signal Unit Construction and Operation

[0040]FIG. 6 is a block diagram in accordance with one preferredembodiment of the present invention depicting a portion of theelectronic circuit for applying the potential across a pair of pins orelectrodes 26 a, 26 b, and for monitoring the current across the pins.

[0041] A computer program executed by a computer system included in thecell culture monitoring and recording system causes a programmable gainamplifier 70 included in a data acquisition board 72 to transmit avoltage representative of that applied across a pair of pins 26 a, 26 binserted into a well for digitization by a measurement input/outputboard 74. The measurement input/output board 74 of the presentembodiment is preferably a MetraByte DAS-8 Data Acquisition and ControlBoard marketed by Keithley Metrabyte Corporation of Taunton, Mass.

[0042] To supply the alternating current voltage that is applied acrossa selected pair of pins 26 a, 26 b, the data acquisition board 72includes a programmable voltage source 76. The programmable voltagesource 76 includes an alternating current generator 78 that produces a370 Hz ±20%, 10 volt peak-to-peak sine wave signal. The output signalproduced by the alternating current generator 78 is transmitted to aprogrammable attenuator 80 also included in the programmable voltagesource 76. Digital excitation level control signals supplied from thecomputer system to the programmable attenuator 80 via excitation levelcontrol signal lines 82 permit adjustment of the peak-to-peak voltagesupplied to a first terminal 84 of a resistor, such as a 20.04 K ohmresistor 86.

[0043] A second terminal 88 of the resistor 86 connects to a bank ofswitches 90. One of the switches 90 is selected by the computer systemfor applying the alternating current voltage supplied by theprogrammable voltage source 76 to a pair of pins 26 a, 26 b that extendinto the well being monitored.

[0044] The AC voltage applied across a pair of pins 26 a, 26 b is alsosupplied to the input of the programmable gain amplifier 70. The gain ofthe amplifier 70 may be adjusted by control signals supplied from thecomputer system via gain control signal lines 92. The output signalsfrom the programmable attenuator 80, and from the programmable gainamplifier 70 are both supplied to a multiplexer 94. Control signalssupplied from the computer system to multiplexer 94 via multiplexercontrol signal lines 96 select one of these three signals forapplication to an input of a sample-and-hold amplifier 100 included inthe measurement input/output board 74.

[0045] The output signal from the sample-and-hold amplifier 100 issupplied to the input of an analog-to-digital converter 102 alsoincluded in the measurement input/output board 74. In addition to beingsupplied to the programmable attenuator 80, the 10 volt peak-to-peakoutput signal from the alternating current generator 78 is also suppliedto the input of a comparator 104. The output signal from the comparator104 changes state each time the alternating current voltage produced bythe alternating current generator 78 passes through zero volts.

[0046] Thus, while the alternating current voltage produced by thegenerator 78 has a potential greater than zero volts, the output signalfrom the comparator 104 is in one state, and while that voltage has apotential less than zero volts, the output signal from the comparator104 is in its other state. The output signal from the comparator 104 issupplied to a programmable timer 106 included in the measurementinput/output board 74.

[0047] As described herein, the voltage present at the second terminal88 of resistor 86 is applied across the two pins of a selected well 12via a switch 90. This “pin voltage”, which is proportional to theconductivity of the medium and the current flow between the two pins, ismeasured by the programmable gain amplifier 70. To efficiently obtain areliable measurement of this voltage (and of the underlying current),the pin voltage is preferably sampled synchronously with the appliedvoltage. This can be done in several ways, two of which are describedbelow.

[0048] 1. Sampling at a selected phase angle. FIG. 7 depicts asinusoidal alternating current waveform 110 for the voltage present atthe output of the alternating current generator 78 together with the adigital waveform 112 of the output signal produced by the comparator104. During initialization of the cell culture monitoring and recordingsystem and at any subsequent time that it is requested by an operator ofthe cell culture monitoring and recording system, the computer programexecuted by the computer system executes a procedure for establishing adelay period (“D”) of a selected duration that is shorter than one cycleof the sine waveform 110. For example, in FIG. 7, the delay periodbegins when the sine waveform 110 is changing from a positive potentialto a negative potential has a potential of zero volts, and ends when thesine waveform 110 has its immediately subsequent maximum positive value.

[0049] In measuring the delay period D, the computer program uses theoutput signal from the comparator 104 in the data acquisition board 72together with the programmable timer 106 included in the measurementinput/output board 74 to determine the duration of one period of thesine waveform 110. The computer program then establishes the delayperiod D at three-fourths of one period of the sine waveform 110. Havingdetermined a proper delay period D, the computer program then loads thatdelay period into the programmable timer 106 so that all subsequentmeasurements of the electrical potential across a pair of pins 26 a, 26b will occur when the voltage supplied to the first terminal 84 of theresistor 86 reaches its maximum value, i.e., at the same selected phaseangle of each cycle.

[0050] In measuring the voltage applied across a pair of pins 26 a, 26b, the programmable timer 106 begins measuring each delay period at theinstant at which the sine waveform 110 is changing from a positivepotential to a negative potential has a potential of zero volts, i.e.immediately after the digital value of the output signal from thecomparator 104 supplied to the programmable timer 106 changes from 0to 1. When the delay period D expires, the programmable timer 106 causesthe sample-and-hold amplifier 100 to sample and hold the voltage of thesignal supplied from the output of the programmable gain amplifier 70via the multiplexer 94 as illustrated in the waveform 114 depicted inFIG. 7.

[0051] The programmable timer 106 also causes the analog-to-digitalconverter 102 to convert the voltage of the analog signal received fromthe sample-and-hold amplifier 100 into a digital form. Subsequently,this digital number is transferred from the measurement input/outputboard 74 to the computer system for storage as raw data suitable forsubsequent analysis and graphic display.

[0052] 2. Sampling at a frequency which is at least an order ofmagnitude greater than the applied voltage. Another way of synchronouslysampling the pin voltage, which is proportional to the current acrossthe electrodes, is to sample and digitize the signal at a frequencywhich is at least an order of magnitude greater than the applied voltage(termed “burst sampling”). In this mode of operation, the pin voltage issampled and digitized a selected number of times (e.g., 10-1000) duringa single cycle of the applied voltage. This can be accomplished, forexample, by triggering the beginning and of storage of a string ofdigitized current values with the rising or falling transition of thedigital waveform 112 of the output signal produced by the comparator104. Such a digitized waveform can be analyzed with respect to theapplied voltage using the computer system to calculate, for example, anyphase lead or lag of the underlying current with respect to the appliedvoltage, as well as the peak-to-peak and/or RMS current values. Thesecurrent values can in turn be used in the calculation of the conductanceof the medium as described herein. An advantage of this approach is thatan accurate estimate of the conductance can be obtained in a singlecycle of the applied voltage, enabling rapid multiplex sampling of aplurality of samples.

[0053] Adjusting Voltage Applied Across a Pair of Pins. In addition toperforming the above, the program executed by the computer system alsodetermines the alternating current voltage to be applied from the secondterminal 88 of the resistor 86 across the pair of pins 26 a, 26 b duringsuch monitoring. To determine this alternating current voltage, thecomputer program first adjusts the programmable attenuator 80 so apotential of approximately 10 millivolts is present at its output and atthe first terminal 84 of the resistor 86. Because the 20.04 K ohmresistance of the resistor 86 separates its first terminal 84 from itssecond terminal 88, and because any cell growth media held in the well12 provides some electrical conductivity between the pair of pins 26 a,26 b inserted therein, initially the voltage at the second terminal 88and across a pair of pin 26 a, 26 b must be less than the value of 10millivolts intended to be used in measuring the conductivity between apair of pins 26 a, 26 b. The computer program then causes themultiplexer 94 to select the output signal from the programmable gainamplifier 70 for application to the input of the sample-and-holdamplifier 100, sets the gain of the programmable amplifier 70 so a peakvoltage of 10 millivolts at the second terminal 88 of the resistor 86will result in the analog-to-digital converter 102 producing a digitalnumber that is approximately 83.3% of the full range of theanalog-to-digital converter 102, and causes the bank of switches 90 toselect a pair of pins 26 a, 26 b for application of the alternatingcurrent voltage.

[0054] The cell culture monitoring and recording system then measuresthe peak alternating current voltage present at the second terminal 88of the resistor 86 that is applied across the pair of pins 26 a, 26 b.If the voltage at the second terminal 88 and across the pair of pins 26a, 26 b is less than 5 millivolts, the computer program doubles thealternating current voltage produced by the programmable attenuator 80repeatedly until the voltage measured at the second terminal 88 exceeds5 millivolts. Having thus applied and measured an alternating currentvoltage across the pair of pins 26 a, 26 b that exceeds 5 millivolts andknowing the setting for the programmable attenuator 80 which producesthat voltage, the computer program then computes a new setting for theprogrammable attenuator 80 that will apply approximately a 10 millivoltalternating current voltage to the second terminal 88 and across thepair of pins 26 a, 26 b, and then transmits control signals setting theattenuator 80 to the computed value.

[0055] Having established the value for the alternating currant voltageapplied by the programmable attenuator 80 to the first terminal 84 ofthe resistor 86, the system is now prepared to monitor and record theelectrical conductivity of the well 12. In measuring the conductivity ofthe well 12, the computer program first repetitively measures thevoltage applied across the pair of pins 26 a, 26 b and at the secondterminal 88 of the resistor 86. For example, applying the method ofmeasuring at a selected phase angle, the computer system collects 16successive values for this voltage, and the computer program thencomputes an average of the 16 values using a box-car filter to obtain asingle, average value for the voltage across the pair of pins 26 a, 26b. Alternatively, applying the burst sampling approach, a single,average value can be obtained from the RMS value of the digitizedsignal. Using the single value of the pin voltage, the value of thevoltage supplied by the programmable attenuator 80 to the first terminal84 of the resistor 86, and the resistance of the resistor 86; thecomputer program then computes the conductivity of the cell growth mediaand cells, if any, between the pair of pins 26 a, 26 b.

[0056] Having determined the conductivity between the pair of pins 26 a,26 b for this well 12, the computer program first stores theconductivity value for subsequent analysis and then proceeds to measurethe conductivity between another pair of pins 26 a, 26 b extending intoanother well 12 in the microwell device 8. In determining theconductivity of each well, the cell culture monitoring and recordingsystem uses the procedures set forth above of first adjusting thealternating current voltage applied across the pair of pins 26 a, 26 b,and then measuring and averaging the voltage applied across the pair ofpins 26 a, 26 b extending into the well 12. This adjusting of theapplied voltage and determining of cell conductivity is repeated overand over until a conductivity has been determined and stored for allwells 12 in the microwell device 8.

[0057] At least one of the wells 12 in the microwell device 8 ispreferably a reference well that holds only cell culture media withoutcells. Furthermore, this reference well must be specifically soidentified to the analysis computer program because that program usesthe conductivity value for the reference well in analyzing theconductivity for all the other wells.

[0058] Analysis of the conductivity of a well that held both cell growthmedia and cells included dividing the conductivity measured for thereference well by the conductivity measured for the well that held boththe cell growth media and cells. Rather than using the electricalconductivity of the reference well as the numerator of a fraction inanalyzing the conductivity of a well that holds both cell growth mediaand cells, it has been found more advantageous in analyzing theconductivity of wells holding both cell growth media and cells tosubtract the conductivity determined for the reference well from theconductivity determined for the well holding both cell growth media andcells. Subtracting the conductivity measured for the reference well,i.e. a well that holds only cell growth media without cells, from theconductivity measured for wells that holds both growth media and cellsremoves the electrical conductivity of the cell growth media from thedata for such wells. Removing the cell growth media conductivity fromthe data for the wells results in data values for the wells holding bothcell growth media and cells that more closely represents the electricalconductivity of only the cells themselves, and the cells' metabolicproducts.

[0059] While the preferred embodiments of the present invention asdescribed above employ a sinusoidal alternating current in monitoringcell cultures, it may be possible to employ any periodic voltagewaveform that is symmetric about zero volts in determining conductivitybetween a pair of pins 26 a, 26 b. Thus, for example, a system formonitoring and recording cell cultures in accordance with the presentinvention could employ an alternating current voltage having atriangular waveform.

[0060] Since a triangular waveform alternating current voltage may beeasily generated using a digital logic circuit, in a system employingsuch a waveform it would be unnecessary to directly measure, asdescribed above, the delay period D. Rather the digital circuits used ingenerating the triangular waveform alternating current voltage couldthemselves directly produce signals for controlling the operation of thesample-and-hold amplifier 100 and the analog-to-digital converter 102.However, such a system for monitoring and recording cell cultures wouldmerely employ a different, well known technique for determining thedelay period for its alternating current voltage that i is equal to aninterval of time between the alternating current voltage having aninstantaneous potential of zero volts and having an instantaneouspotential equal to the maximum voltage of the alternating currentvoltage.

[0061] Although the invention has been described with respect toparticular embodiments and features, it will be appreciated that variouschanges and modifications can be made without departing from theinvention. As an example, and in another preferred embodiment, theprobes are inserted into the well from the bottom of the well to allowfor easy sterilization of the unit and minimal media volume.

It is claimed:
 1. High throughput screening apparatus comprising asubstrate defining a plurality of discrete microwells on a substratesurface, at a well density of greater than about 100/cm², where the wellvolumes are such as to accommodate at most about 10³ cells/well, meansfor measuring the conductance in each microwell, said means including(i) a pair of electrodes adapted for insertion into a well on thesubstrate, and (ii) signal means for applying a low-voltage, AC signalacross said electrodes, when the electrodes are submerged in suchmedium, and for synchronously measuring the current across theelectrodes, to monitor the level of growth or metabolic activity ofcells contained in each well.
 2. The apparatus of claim 1, wherein saidsignal means is effective to generate a signal whose peak-to-peakvoltage is between 5 and 10 mV.
 3. The apparatus of claim 2, whereinsaid signal means includes feedback means for adjusting the signalvoltage level to a selected peak-to-peak voltage between 5 and 10 mV. 4.The apparatus of claim 1, wherein said signal means is designed tosample the voltage of the applied signal at a selected phase angle ofthe signal.
 5. The apparatus of claim 1, wherein said signal means isdesigned to sample the voltage of the applied signal at a frequencywhich is at least an order of magnitude greater than that of saidsignal.
 6. The apparatus of claim 1, wherein said wells are adapted tohold at most between 1-100 cells/well.
 7. A high throughput screeningmethod comprising placing cells on a substrate defining a plurality ofdiscrete microwells on a substrate surface, at a well density of greaterthan about 100/cm², with the number of cells in each well being lessthan about 10³, where the cells in each well have been exposed to aselected agent, and determining the conductance in each well, byapplying a low-voltage, AC signal across a pair of electrodes placed inthat well, and synchronously measuring the conductance across theelectrodes, to monitor the level of growth or metabolic activity ofcells contained in each well.
 8. The method of claim 7, wherein thesignal applied across the electrodes is between 5 and 10 mV.
 9. Themethod of claim 7, wherein said measuring includes measuring theconductance in the medium at a selected phase angle of the signal. 10.The method of claim 7, wherein said measuring is designed to measure theconductance of the medium at a measuring frequency which is at least anorder of magnitude greater than that of said signal.
 11. The method ofclaim 7, wherein the wells contain at most 10-100 cells.